CN104254599B - Using the biological protection of Lactobacillus paracasei bacterial strain - Google Patents

Using the biological protection of Lactobacillus paracasei bacterial strain Download PDF

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CN104254599B
CN104254599B CN201380018785.9A CN201380018785A CN104254599B CN 104254599 B CN104254599 B CN 104254599B CN 201380018785 A CN201380018785 A CN 201380018785A CN 104254599 B CN104254599 B CN 104254599B
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lactobacillus
bacterial strain
lactobacillus paracasei
food product
product
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CN104254599A (en
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蒂娜·霍恩贝克
M·利斯贝格
S·K·迪默
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Section Hansen Co ltd
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Priority claimed from PCT/EP2012/056384 external-priority patent/WO2012136830A1/en
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
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    • A23V2400/165—Paracasei
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Abstract

The present invention relates to biological protection field, more particularly to the Lactobacillus paracasei CHCC14676 bacterial strains that deposit number is DSM25612.Moreover, the present invention relates to a kind of antifungal composition comprising the bacterial strain, a kind of antifungal composition, the food product comprising such a antifungal composition, feed product and medicine comprising the bacterial strain and at least one Lactobacillus rhamnosus strain, a kind of method for manufacturing such a food product, feed product and medicine, a kind of reduction such a food product, feed product and the fungi of medicine and the method for mould bacterial content and the purposes of the antifungal composition.

Description

Using the biological protection of Lactobacillus paracasei bacterial strain
Technical field
The present invention relates to biological protection field, more particularly to the Lactobacillus paracasei that deposit number is DSM25612 (Lactobacillus paracasei) CHCC14676 bacterial strains.Moreover, the present invention relates to a kind of anti-true comprising the bacterial strain Bacteria composition, one kind include the bacterial strain and at least one Lactobacillus rhamnosus (Lactobacillus rhamnosus) bacterial strain Antifungal composition, food product, feed product and medicine comprising such a antifungal composition, one kind manufactures such a food The method of product, feed product and medicine, a kind of fungi and mould bacterial content for reducing such a food product, feed product and medicine Method, and the purposes of the antifungal composition.
Technical background
For many years, lactic acid bacteria is used for the storage life for extending food product always.Lactic acid bacteria produces breast during the fermentation Acid and other organic acids, so as to reduce the pH value of the food product, make them unsuitable for such as pathogenetic bacteria, yeast and fungi The growth of unwanted microorganism.
In addition, some lactic acid bacterias also produce the metabolin with antimicrobial acivity.
U.S. Patent application US2011/0045134 is related to Lactobacillus paracasei and assigns antifungal property to fermented dairy product Purposes.
European patent application EP1442113 is related to propionibacterium jensenii (Propionibacterium jensenii) and all Such as the mixture of the kind (Lactobacillus sp.) of the lactobacillus of Lactobacillus paracasei, it has antimicrobial acivity, is used for Biological protection.
But, it is still desirable to there is the anti-true of improvement as single bacterial strain or with what other biological protectiveness bacterial strain was combined The Bioprotective of bacterium effect.
The content of the invention
It is an object of the invention to provide can be used as Bioprotective to have efficient novel lactic acid bacteria strains.
The present inventor is by largely screening and studying the biological protection culture it has been found that with the market commercialization Compare, some Lactobacillus paracasei bacterial strain confrontation yeast and the significantly higher effect of mould.
The present inventor is it has been further discovered that a certain group of lactic acid bacteria shows significant association when being combined with another group of lactic acid bacteria Same anti-microbial effect., it is surprising that the anti-microbial effect of this combined two groups of bacteriums is more than this two groups of bacteriums lists The summation of only effect.
Brief description of the drawings
Fig. 1 shows growth of the mould in the Yoghourt containing 1.5% fat, and the Yoghourt is individually by starter culture YF- L901 (top row), by starter culture YF-L901 together with HOLDBACTMYM-B (center row) and by starter culture YF-L901 Fermented together with Lactobacillus paracasei CHCC14676.Target contaminant is from left to right added with the concentration referred in text respectively：It is short It is close mould (Penicillium brevicompactum) (M1), penicillium communne (Penicillium commune) (M6), variegated Aspergillus (Aspergillus versicolor) (M7) and light spore mould (Penicillium glabrum) (M8).The Yoghourt It has been incubated at 7 ± 1 DEG C 61 days.
Fig. 2 shows the inferior Dbaly yeast (Debaryomyces of the Chinese added into the Yoghourt containing 1.5% fat Hansenii the cell count of bacterium) is separated, the Yoghourt is individually by starter culture YF-L901 (reference) or by starter culture YF-L901 is together with HOLDBACTMYM-B cultures (HoldBac YM-B), by starter culture YF-L901 together with HOLDBACTM YM-C cultures (HoldBac YM-C), or by starter culture YF-L901 together with Lactobacillus paracasei bacterial strain CHCC14676 (14676) ferment together.Yoghourt is stored in 7 ± 1 DEG C and analyzed in reasonable time interval.
Fig. 3 shows growth of the mould on flat board, the flat board be prepared in individually by starter culture (reference, first Width figure), by starter culture together with Lactobacillus paracasei CHCC14676 (the second width figure), by starter culture together with rhamnose Lactobacillus CHCC5366 (the 3rd width figure), or by starter culture together with Lactobacillus paracasei CHCC14676 and rhamnose breast bar The milk of bacterium CHCC5366 combination (the 4th width figure) fermentation.A left side is moved to the concentration referred in text clockwise from the upper left of flat board It is lower to add target contaminant respectively：Receive ground mould (Penicillium nalgiovense), penicillium communne (Penicillium Commune), aspergillus versicolor (Aspergillus versicolor) and skin fall mould (Penicillium crustosum).Institute Flat board is stated to be incubated at 7 ± 1 DEG C 12 days.
Fig. 4 shows growth of the mould on flat board, the flat board be prepared in individually by starter culture (reference, first Width figure), by starter culture together with Lactobacillus paracasei CHCC14676 (the second width figure), by starter culture together with rhamnose Lactobacillus CHCC14226 (the 3rd width figure) or by starter culture together with Lactobacillus paracasei CHCC14676 and rhamnose breast bar The milk of bacterium CHCC14226 combination (the 4th width figure) fermentation.Moved to clockwise from the upper left of flat board with the concentration referred in text Target contaminant is added respectively in lower-left：Receive ground mould (Penicillium nalgiovense), penicillium communne (Penicillium Commune), aspergillus versicolor (Aspergillus versicolor) and skin fall mould (Penicillium crustosum).Institute Flat board is stated to be incubated at 7 ± 1 DEG C 12 days.
Fig. 5 shows growth of the mucor (Mucor ssp.) in Greek style Yoghourt (Greek yoghurt), described uncommon Cured formula Yoghourt is prepared in the milk (left side cup) only to be fermented by starter culture, or is done in Lactobacillus rhamnosus CHCC5366 and pair Lactobacillus paracasei CHCC14676 all in the presence of the milk (right side cup) that ferments.
Fig. 6 shows growth of the rhizopus stolonifer (Rhizopus stolonifer) in Greek style Yoghourt, the Greek style Yoghourt is prepared in only by starter culture (R, i.e. reference), by starter culture and HOLDBACTMYM-B (YM-B), is trained by bottle opener Thing and Lactobacillus rhamnosus CHCC5366 (5366) are supported, or by starter culture and Lactobacillus rhamnosus CHCC5366 and secondary cheese The milk of lactobacillus CHCC14676 combination (5366+14676) fermentation.
Fig. 7 shows penicillium brevicompactum (M1), penicillium communne (M6), aspergillus versicolor (M7) and light spore mould (M8) in sour cream In growth, the sour cream is prepared in only by starter culture (top go)；By starter culture and HOLDBACTMYM-B (in Between row)；Or combination (the bottom by starter culture and Lactobacillus paracasei CHCC14676 and Lactobacillus rhamnosus CHCC12697 OK) the milk of fermentation.
Fig. 8 shows growth of the inferior Dbaly yeast of the Chinese in sour cream, and the sour cream is prepared in only by bottle opener culture Thing (reference)；By starter culture and HOLDBACTMYM-B(YM-B)；Or by starter culture, Lactobacillus paracasei The milk of CHCC14676 and Lactobacillus rhamnosus CHCC12697 (12697+14676) fermentations.
Fig. 9 shows saccharomyces cerevisiae (Saccharomyces cerevisiae) in white halogen cheese (white brine Cheese the growth on), the white halogen cheese are prepared in only by starter culture (reference)；By starter culture and HOLDBACTM YM-B(YM-B)；Or by starter culture, Lactobacillus paracasei CHCC14676 and Lactobacillus rhamnosus CHCC5366 (5366+ 14676) milk of fermentation.
Figure 10 shows life of the kluyveromyces marxianus (Kluyveromyces marxianus) on white halogen cheese Long, the white halogen cheese is prepared in only by starter culture (reference)；By starter culture and HOLDBACTMYM-B(YM-B)； Or by starter culture, Lactobacillus paracasei CHCC14676 and Lactobacillus rhamnosus CHCC5366 (5366+14676) fermentation Milk.
Figure 11 shows growth of the penicillium communne on white halogen cheese, and the white halogen cheese is prepared in only by starter culture (R, i.e. reference)；By starter culture and HOLDBACTMYM-B(YM-B)；Or by starter culture, Lactobacillus paracasei The milk of CHCC14676 and Lactobacillus rhamnosus CHCC5366 (5366+14676) fermentations.
Figure 12 shows life of the mould Pan Shi moulds (Penicillium paneum) in the Yoghourt containing 1.5% fat Long, the Yoghourt is prepared in by single starter culture (R, i.e. reference), by starter culture and HOLDBACTM YM-B(YM- B), by starter culture and Lactobacillus rhamnosus CHCC5366 (5366), or by starter culture and Lactobacillus rhamnosus The milk that CHCC5366 and Lactobacillus paracasei CHCC14676 combination (5366+14676) is fermented.
Figure 13 shows that mould skin falls growth of the mould in the Yoghourt containing 1.5% fat, and the Yoghourt is prepared in by list Only starter culture (top row), by starter culture and HOLDBACTMYM-B (the second row), by starter culture and rhamnose Lactobacillus CHCC5366 (the third line), or by starter culture and Lactobacillus rhamnosus CHCC5366 and Lactobacillus paracasei The milk of CHCC14676 combination (last column) fermentation.Target contaminant is added with the concentration of 100 spore/cups, and it is described Yoghourt is incubated 36 days in 7 ± 1 DEG C (left columns), 12 ± 1 DEG C (intermediate hurdles) or 22 ± 1 DEG C (right column).
Embodiment
Definition
As used herein, term " lactic acid bacteria " refers to gram-positive micro- aerobic or anaerobism bacterium, and its sugar fermentation produces Acid, including lactic acid are main caused acid.Industrial most useful genus lactubacillus are in " lactobacillus (Lactobacillales) " Mesh, it includes lactococcus (Lactococcus spp.), streptococcus (Streptococcus spp.), lactobacillus (Lactobacillus spp.), Leuconostoc (Leuconostoc spp.), pseudo- Leuconostoc (Pseudoleuconostoc spp.), Mycosphaerella (Pediococcus spp.), brevibacterium (Brevibacterium ) and enterococcus spp (Enterococcus spp.) spp..These bacteriums often combine individually or with other lactic acid bacterias and are used as eating Product culture.
Term " food " is intended to also include cheese.Term " cheese " is interpreted to embrace any cheese, including hard cheese, Semi-rigid cheese and soft cream, such as following types of cheese：Ka Teji cheese (Cottage), phenanthrene reach cheese (Feta), cut Up to cheese (Cheddar), Palma cheese (Parmesan), cheese (Mozzarella), angstrom door tal fibre cheese do not draw in Surrey (Emmentaler), red rich cheese (Danbo), up to cheese (Gouda), Ai Danmu cheese (Edam), Fei Dashi cheese (Feta-type), blue cheese (blue cheeses), halogen cheese (brine cheese), card awns Bel cheese (Camembert) With cheese in cloth (Brie).Skilled in the art realises that how coagulation is transformed into cheese, method can be found from document, See, for example, Kosikowski, F.V and V.V.Mistry, " Cheese and Fermented Milk Foods ", 1997, Three editions, F.V.Kosikowski, L.L.C.Westport, CT. is as used herein, will have dense less than 1.7% (w/w) NaCl The cheese of degree is referred to as " less salt cheese ".
The lactic acid bacteria for being typically included the bacterium of lactobacillus species and milk-globule strain is supplied to dairy industry, itself or with freezing Or the form of lyophilized culture is bred for batch bottle opener, or with so-called " direct putting type " (" Direct Vat Set ") (DVS) Culture form, it is intended to be directly inoculated into and be used for the breast system for producing such as fermented-milk products or cheese in round or fermenter Product.Such a lactic acid bacteria culture is commonly referred to as " starter culture " or " bottle opener ".
Term " mesophile " herein refers to grows most vigorous microorganism under moderate temperature (15 DEG C to 40 DEG C).Industry Upper most useful mesophili bacteria includes lactococcus and Leuconostoc.Term " mesophilic digestion " herein refers at about 22 DEG C Fermentation at a temperature of about 35 DEG C.Term " mesophilic digestion dairy products " refers to the mesophilic digestion institute by medium temperature starter culture The fermented-milk products of preparation, and including such as buttermilk, yogurt (sour milk), cultured milk (cultured milk), Si Mei The fermentation of tower that sour cream (smetana), sour cream and fresh cheese (such as quark cheese, tvarog cheese and cream cheese) Dairy products.
Term " Thermophilic Bacteria " herein refers in the most vigorous microorganism of the temperature growth higher than 43 DEG C.It is industrial most useful Thermophilic bacteria include streptococcus and lactobacillus.Term " hot fermentation " herein refers to higher than about 35 DEG C (such as About 35 DEG C to about 45 DEG C) at a temperature of fermentation.Term " hot fermentation dairy products " refers to by thermophilic starter culture Fermented-milk products prepared by hot fermentation, and including such as hair of solidification type yoghourt, stirred yoghurt and drinking yoghourt Ferment dairy products.
Term " milk " should be understood by entering to any mammal of such as milk cow, sheep, goat, buffalo or camel The galactosis thing of residual that row is milked and obtained.In a preferred embodiment, milk is milk.Term milk also includes former by plant Expect the protein/fat solution of manufacture, such as big soymilk.
Term " milk substrate " can be can the method according to the invention experience fermentation any raw milk's raw material and/or processing Milk material.Therefore, useful milk substrate includes but is not limited to solution/suspension of any milk comprising protein or class dairy products, Such as whole milk or low fat milk, defatted milk, buttermilk, recovery milk powder, condensed milk, dried milk, whey, whey penetrant, lactose, lactose Crystalline mother solution, whey protein concentrate or cream.Obviously, milk substrate is probably derived from any mammal, for example, as basic Upper pure mammal milk restores milk powder.
Before fermentation, milk substrate can be homogenized according to procedures known in the art and pasteurization.
As used herein, " homogenize " and mean and be thoroughly mixed to obtain solvable suspension or emulsion.If fermenting Homogenized before, then its purpose is to which milk fat is broken for into less size, it is no longer separated with milk.This can To be realized by forcing milk under high pressure through aperture.
As used herein, " pasteurization ", which is meant, is handled milk substrate, to reduce or eliminate such as microorganism Living Organism presence.Preferably, pasteurization is by maintaining set period to realize assigned temperature.It is described to specify Temperature is generally realized by heating.Temperature and duration can be selected, to kill or inactivate some bacteriums, such as it is harmful thin Bacterium.Next quick cooling step can be carried out.
In the method for the present invention, " fermentation " mean by the effect of microorganism by it is carbohydrate-modifying be alcohol or Acid.Preferably, it is lactic acid that the fermentation in method of the invention, which is included Lactose conversion,.
Be intended for use in dairy products production in fermentation process be it is well known that and those skilled in the art will be appreciated that such as What suitable process conditions of selection, such as temperature, oxygen, the quantity and characteristic of microorganism, and process time.Obviously, to hair Ferment condition carry out selection be in order to contribute to the present invention realization, i.e., in order to obtain the dairy products of solid-state form (such as cheese) or The dairy products (such as fermented-milk products) of liquid form.
The present invention composition offered the advantage that, such as on food product, feed product and medicine and In the mankind and mammal, the unwanted microorganism selected from fungi, bacterium and its mixture can be suppressed.Especially expected resistance Only and/or suppress such as yeast and mould fungi growth.Therefore, in a preferred embodiment, term " resists micro- life Thing " should be understood " antimycotic ".
Term " unwanted microorganism " herein refers to pathogenic microorganism and/or can make food product, feed product Or the microorganism that medicine is rotten, such as bacterium and fungi, such as yeast.The present invention composition offered the advantage that, example Such as on food product, feed product and medicine and in the mankind and mammal, selected from fungi, bacterium and its mixture Unwanted microorganism can be suppressed.The growth of the fungi of especially expected prevention and/or suppression such as yeast and mould.
The term " suppression " related to yeast and mould is meant, for example, the growth of yeast and mould or quantity or dense Spend in the food product for example comprising the bacterial strain according to the present invention and/or on its surface than in the food not comprising such bacterial strain It is low in product product and/or on its surface.
Herein, term " mutant " should be understood that for example, by genetic engineering, radiation and/or chemical treatment hand Bacterial strain derived from section from the bacterial strain of the present invention.Preferably, the mutant is a kind of mutant of functional equivalent, for example, with mother Bacterial strain has the mutant of characteristic (such as on antifungal property) that is substantially the same or improving.Such mutant is this A part for invention.Especially, term " mutant " refers to by making the bacterial strain of the present invention undergo at any conventional use of mutagenesis The bacterial strain managed and obtained, or spontaneous caused mutant, the mutagenic treatment include using such as ethyl methane sulfonate (EMS) or N- first The chemical mutagen processing of base-N'- nitro-N-nitroguanidines (NTG), with UV light processings.Mutant may experienced mutagenesis for several times Handle (single treatment should be understood that a mutagenesis steps and subsequent screening/selection step), still, it is presently preferred to carry out No more than 20 times, or no more than 10 times, or no more than 5 times processing (or screening/selection step).In a currently preferred mutation In body, compared to mother strains, 5%, or less than 1% is less than in its bacterial genomes, or the nucleotides even less than 0.1% is by quilt Another nucleotides substitution, or lacked.
Contrary is explicitly indicated that unless otherwise indicated herein or by context, in the context of the explanation present invention Term " a (one/mono-/a kind of) " that (particularly in the context of claim) uses, " an (one/mono-/a kind of) " and " should/described " and similar term should be understood that and cover odd number and plural number.Unless otherwise indicated, term "comprising", " tool Have ", " comprising " and " containing " be construed as open-ended term (representing " including but is not limited to ").Unless separately have herein Bright, number range described herein is provided merely as being individually recited the simple statement side for each discrete values for falling into the scope Method, and each discrete values are incorporated into specification as being individually recited in the text.Unless separately have herein Illustrate or contrary is explicitly indicated that by context, all methods described herein can be in any suitable order Implement.Unless otherwise stated, provided herein is any and all embodiment or citing language (such as " such as ") use It is intended merely for that the present invention is better described, not as limiting the scope of the present invention.All language in specification The element that shall not be construed as any undesired protection is necessary for implementing the present invention.
Embodiment of the present invention and aspect
The present inventor is to Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus paracasei and rhamnose 200 candidate strains of lactobacillus are screened, to find the most effective of the wide scope microorganism of confrontation such as yeast and mould Single bacterial strain or double bacterial strains combination.
Screening is carried out in the matrix based on milk with simulating the model analysis of mesophilic digestion dairy products as far as possible, the matrix In added with containing or not contain the related starter culture of biological protection candidate, and the matrix with mesophilic digestion It is fermented under conditions of dairy products correlation.Target organs are isolated from medium temperature fermented-milk products.Using below both as Benchmark：From Denis company of Denmark (Danisco A/C, Denmark) HOLDBACTMThe lactic acid bacteria of culture purifying, Yi Jihan There are lactic acid bacteria and the complete HOLDBAC both bacterium acidi propioniciTMYM-B and HOLDBACTMYM-C cultures.
The model analysis is described in european patent application EP11161609.0.
When detecting for 25 DEG C, find to have in Lactobacillus paracasei and Lactobacillus rhamnosus 17 kinds of candidate strains usually can be with The benchmark lactic acid bacteria considerably or compared with it more preferably suppresses 12 kinds of instruction fungies.
When being detected on Yoghourt, a bacterial strain of Lactobacillus paracasei, i.e., Germany is preserved in deposit number DSM25612 Microorganism and the Lactobacillus paracasei bacterial strain CHCC14676 of Cell Culture Collection (DSMZ), suppressing yeast and mould Aspect is shown than commercially available biological protection culture significantly more preferably.
As the inhibition of bacterial strain confrontation yeast and mould can be described by the embodiment in following article, pass through by Fermented-milk products store one section of suitable storage time to determine at a suitable temperature.
In general, the suitable temperature of this method should be implemented depending on specific food product, feed product or medicine lead to The temperature for being often stored and/or manufacturing.The temperature of the product is generally stored between 5 DEG C and 26 DEG C, it is preferable that the temperature It is about 8 DEG C.
Storage time at said temperatures depends on the time that the food product, feed product or medicine generally store (storage life).Storage time is typically 5-65 days, preferably 7-60 days, more preferably 7-28 days, and even further preferably, the storage Time is about 21 days, and more preferably up to 60 days or more.
Therefore, the first aspect of the present invention is related to Lactobacillus paracasei bacterial strain, and it is selected from the group consisted of：With preservation Numbering DSM25612 is preserved in the Lactobacillus rhamnosus strain of German microorganism and Cell Culture Collection (DSMZ) CHCC14676, and by its derivative mutant strain.
Therefore, in addition to preservation strain mentioned above, the present invention also relates to the mutant derived from this bacterial strain, That is, described mutant is obtained by using the preservation strain CHCC14676 for parent material.The mutant strain can be such as The preservation strain is derived from by following means：Genetic engineering, radiation, UV light, chemical treatment and/or induced gene group change Method.It will be suppressed according to mutant of the present invention and/or prevent some bacteriums or fungi, the growth of preferred mould.It is excellent Selection of land, when in analysis as described in Example 1, using its growth by repressed penicillium brevicompactum, penicillium communne, variegated One of mould of the inferior Dbaly yeast of aspergillus, the Chinese or light spore mould as be measured with reference to organism when, the mutant and Its mother strains, which is compared, has substantially at least 80% or more, at least 90% or more, at least 95% or more, or even as high as 100% or more anti-mycotic efficiency.
It will be apparent to those skilled in the art that be parent material by using the bacterial strain of the preservation, skilled reader by Conventional mutagenesis or again isolation technics can routinely obtain keep correlated characteristic described herein and advantage enter one Mutant of step or derivatives thereof.Therefore, the term of first aspect is related to by using described " by its derivative mutant strain " The mutant strain that the bacterial strain of preservation is obtained by parent material.
Second aspect is related to a kind of antifungal composition, and it does comprising at least one pair according to the first aspect of the invention Lactobacillus paracasei bacterial strain, preferably Lactobacillus paracasei bacterial strain CHCC14676.
When from different Lactobacillus rhamnosus strain combined test Lactobacillus paracasei bacterial strain CHCC14676, exceed to anticipate Material ground finds that these are combined even than single any bacterial strain more preferably, in the case of the total concentration identical of cell.One In individual embodiment, the present invention relates to Lactobacillus paracasei bacterial strain CHCC14676's and Lactobacillus rhamnosus strain CHCC5366 Combination.In another embodiment, the present invention relates to Lactobacillus paracasei bacterial strain CHCC14676 and Lactobacillus rhamnosus strain CHCC14226 combination.Also in another embodiment, the present invention relates to Lactobacillus paracasei bacterial strain CHCC14676 and mouse Lee's sugar lactobacillus strain CHCC12697 combination.These combinations seem than the benchmark culture from Denis company of Denmark HOLDBACTMYM-B and HOLDBACTMYM-C is more effective.
Therefore, in a preferred embodiment, it is related to the present invention relates to antimicrobial compositions and more preferably anti-true Bacteria composition, the composition includes is preserved in German microorganism and Cell Culture Collection with deposit number DSM25612 (DSMZ) Lactobacillus paracasei bacterial strain CHCC14676 or its derivative mutant, and at least one Lactobacillus rhamnosus bacterium Strain.Preferably, at least one Lactobacillus rhamnosus strain is selected from the Lactobacillus rhamnosus for DSM24616 by deposit number Lactobacillus rhamnosus strain CHCC14226 that bacterial strain CHCC12697, deposit number are DSM24652, deposit number are DSM23035 Lactobacillus rhamnosus strain CHCC5366, and the group that the mutant strain derived from these preservation strains forms.
The present invention in one aspect thus a kind of antimicrobial compositions and more preferably antifungal composition are provided, it is described Composition includes Lactobacillus paracasei bacterial strain CHCC14676 or its derivative mutant, and Lactobacillus rhamnosus strain CHCC14226 or its derivative mutant.On the other hand, the present invention provides a kind of antimicrobial and more preferably antimycotic group Compound, it includes at least Lactobacillus paracasei bacterial strain CHCC14676 or by its derivative mutant, and Lactobacillus rhamnosus bacterium Strain CHCC5366 or by its derivative mutant.In yet a further aspect, the present invention provides a kind of antimicrobial compositions and more excellent Antifungal composition is selected, the composition includes at least Lactobacillus paracasei bacterial strain CHCC14676 or its derivative mutant, with And Lactobacillus rhamnosus strain CHCC12697 or its derivative mutant.Preferably, the secondary cheese in the present composition The combination of lactobacillus strain and the Lactobacillus rhamnosus strain cooperates with terms of their antimicrobial and/or antifungal activity Effect.
The antifungal composition typically comprises the bacterium of conc forms, including the concentrate for freezing, drying or freezing, The viable cell concentrations that the bacterium of the conc forms typically has are in every gram of composition 104To 1012Cfu (colony forming unit) Scope, including every gram of composition at least 104Cfu, such as at least 105Cfu/g, for example, at least 106Cfu/g, such as at least 107Cfu/g, for example, at least 108Cfu/g, such as at least 109Cfu/g, for example, at least 1010Cfu/g, such as at least 1011cfu/g。 Therefore, composition of the invention preferably to freeze, dry or lyophilized form exist, for example, as direct putting type (DVS) culture. However, as used herein, the composition is also likely to be to be suspended in the freezing, the cell concentration thing dried or freezed such as The liquid obtained after in the fluid matrix of water or PBS.When the composition of the present invention is suspension, the concentration of living cells In every milliliter of composition 104To 1012Cfu (colony forming unit) scope, including every milliliter of composition at least 104Cfu, such as At least 105Cfu/ml, for example, at least 106Cfu/ml, such as at least 107Cfu/ml, for example, at least 108Cfu/ml, such as at least 109Cfu/ml, for example, at least 1010Cfu/ml, such as at least 1011cfu/ml。
The composition can contain antifreezing agent and/or conventional additives as further composition, the conventional additives Nutrition including such as yeast extract, sugar and vitamin (such as vitamin of vitamin A, C, D, K or vitamin B complex) Element.Can be added to the present invention composition in suitable antifreezing agent be improve microorganism cold resistance composition, such as sweet dew Alcohol, sorbierite, sodium tripolyphosphate, xylitol, glycerine, gossypose, maltodextrin, erythrite, threitol, trehalose, grape Sugar and fructose.Other additives may include, such as carbohydrate, flavor enhancement, mineral matter, enzyme (such as rennet (rennet), lactase and/or phosphatidase).
In the composition of the invention comprising Lactobacillus paracasei bacterial strain CHCC14676 and Lactobacillus rhamnosus strain, Ratio between Lactobacillus paracasei bacterial strain CHCC14676 and the Lactobacillus rhamnosus strain, such as Lactobacillus paracasei are thin Ratio between the concentration or quantity of bacterium and the concentration or quantity of Lactobacillus rhamnosus bacterium, preferably 1:100 to 100:1, it is excellent Elect 1 as:10 to 10:1.
The antifungal composition of the present invention can be with being easily degraded by microorganisms and/or easily by times of yeast and mould contamination What food product, feed product and medicine are used in connection with.These include but is not limited to fruits and vegetables (including derived product), paddy Thing and cereal derived product, dairy products, meat, poultry and marine products class.In particularly preferred embodiments, the composition It is used in connection with dairy products and/or meat and poultry.In a preferred embodiment, composition of the invention is used for all Additive is used as in such as preparation of Yoghourt, tvarog cheese, sour cream, cream cheese, white halogen cheese dairy products.
In a preferred embodiment, composition of the invention is used for antimycotic, such as yeast and mould.This meaning Taste the composition and is used to suppress and/or prevent during dairy processing industry, especially in milk fermentation process, causes to pollute Fungi growth.The composition of the present invention can be used in, such as suppress and/or prevent the growth of yeast, such as with subordinate's Yeast：Kluyveromyces (Klyveromyces) (such as kluyveromyces marxianus (K.marxianus), Kluyveromyces Lactis dimension Yeast (K.lactis)), pichia (Pichia) (such as fermentation Pichia pastoris (P.fermentans)), Ye Shi saccharomyces (Yarrowia) (such as Yarrowia lipolytica (Y.lipolytica)), candida (Candida) (such as pure mellow wine vacation silk ferment Female (C.sake)) etc.；Or suppress and/or prevent the growth of mould, such as from the mould of subordinate：Penicillium (Penicillium) (such as receive ground mould, penicillium communne, skin fall mould, penicillium brevicompactum, light spore mould), mucor (Mucor Spp.), cladosporium category (Cladiosporium ssp.), aspergillus (Aspergillus) (such as aspergillus versicolor), De Bali ferment Mother's category (Debaryomyces) (such as inferior Dbaly yeast of the Chinese (D.hansenii)) etc..It is particularly preferred that use the present invention Composition suppress and/or prevent the growth such as sowed：Kluyveromyces marxianus, Yarrowia lipolytica, receive ground mould, branch spore Pseudomonas, penicillium communne, mucor, penicillium brevicompactum, aspergillus versicolor, skin fall mould, Kluyveromyces lactis and/or Han Xunde Balis Yeast.
Antifungal composition according to the second aspect of the invention is also used as medicine, and it is true to be preferred for treatment pathogenicity Bacterium infects, such as medicine of pathogenicity yeast infection.
In the third aspect, the present invention relates to comprising Lactobacillus paracasei bacterial strain according to the first aspect of the invention or according to Food product, feed product or the medicine of the antifungal composition of the second aspect of the present invention.
In a preferred embodiment, the food product, feed product or medicine are food product.
In a further preferred embodiment, such a food product be selected from by fruit and fruit derived product, vegetables and What vegetables derived product, cereal and cereal derived product, dairy products, meat, poultry and marine products category and its mixture formed Group.
In one even more preferably embodiment, the food product is dairy products, preferably mesophilic digestion dairy products Or hot fermentation dairy products, such as fresh cheese, Yoghourt, sour cream or tvarog cheese.
In a further preferred embodiment, the food product is meat or poultry.
In a preferred embodiment, the food product, feed product or medicine are medicines.
Preferably, the medicine is to can be used for applying human or animal antimycotic combination according to the second aspect of the invention Thing, with the product for suppressing pathogenic microbes and mitigating the symptom related to pathogenic microbes.The example of such a symptom includes The symptom related to yeast infection.In this kind of embodiment, the medicine can be the unit dose for including antifungal composition Type.Preferably, the unit dosage forms are capsule or tablet.However, the unit dosage forms can also be suitable for being administered to mucous membrane or Skin, and therefore exist in the form of paste, emulsifiable paste, ointment etc..
The fourth aspect of the present invention is related to a kind of food product being used to manufacture according to the third aspect of the invention we, feed production The method of product or medicine, methods described, which is included in the manufacturing process of the food product, feed product or medicine, adds at least A kind of Lactobacillus paracasei bacterial strain according to the first aspect of the invention or antimycotic combination according to the second aspect of the invention Thing.Preferably, methods described is additionally included in manufacturing process the step of controlling Fabrication parameter, described at least one secondary dry to cause The concentration of Lactobacillus paracasei bacterial strain remains constant or improved.
In a preferred embodiment, the concentration of at least one Lactobacillus paracasei bacterial strain is produced for the food At least the 1 × 10 of product, feed product or medicine6At least the 1 of cfu/g or respectively described food products, feed product or medicine ×106Cfu/ml, or at least the 1 × 10 of the surface of respectively described food product, feed product or medicine5cfu/cm2.It is preferred that Ground, the concentration of at least one Lactobacillus paracasei bacterial strain is the food product, feed product or medicine at least 5 × 106At least the 5 × 10 of cfu/g or respectively described food products, feed product or medicine6Cfu/ml, or respectively described food At least the 5 × 10 of the surface of product, feed product or medicine5cfu/cm2, it is such as the food product, feed product or medicine At least 1 × 107At least the 1 × 10 of cfu/g or respectively described food products, feed product or medicine7Cfu/ml, or each For at least the 1 × 10 of the surface of the food product, feed product or medicine6cfu/cm2, it is such as the food product, feed At least the 5 × 10 of product or medicine7At least the 5 × 10 of cfu/g or respectively described food products, feed product or medicine7cfu/ Ml, or at least the 5 × 10 of the surface of respectively described food product, feed product or medicine6cfu/cm2。
When by addition include Lactobacillus paracasei bacterial strain CHCC14676 or its derivative mutant and at least one sandlwood When sugared lactobacillus strain is to manufacture the food product, feed product or medicine, Lactobacillus paracasei bacterial strain CHCC14676 or its The concentration of derivative mutant and/or the concentration of at least one Lactobacillus rhamnosus strain be respectively the food product, At least the 1 × 10 of feed product or medicine6Cfu/g or respectively described food products, feed product or medicine at least 1 × 106Cfu/ml, or at least the 1 × 10 of the surface of respectively described food product, feed product or medicine5cfu/cm2.Preferably, The concentration and/or at least one Lactobacillus rhamnosus of Lactobacillus paracasei bacterial strain CHCC14676 or its derivative mutant The concentration of bacterial strain is respectively at least the 5 × 10 of the food product, feed product or medicine6Cfu/g or respectively described food At least the 5 × 10 of product, feed product or medicine6Cfu/ml, or the table of respectively described food product, feed product or medicine At least the 5 × 10 of face5cfu/cm2.In a further embodiment, Lactobacillus paracasei bacterial strain CHCC14676 or its spread out The concentration of raw mutant and/or the concentration of at least one Lactobacillus rhamnosus strain are respectively the food product, feeding Expect at least the 1 × 10 of product or medicine8Cfu/g or respectively described food products, feed product or medicine at least 1 × 108Cfu/ml, or at least the 1 × 10 of the surface of respectively described food product, feed product or medicine7cfu/cm2。
In a preferred embodiment, Fabrication parameter is controlled in the fabrication process so that Lactobacillus paracasei bacterial strain The concentration of CHCC14676 (or its derivative mutant) and at least one Lactobacillus rhamnosus strain improves or kept permanent It is fixed.
It is most readily by mixing with the food product, feed product or medicine that can be blended according to the antifungal composition of the present invention Close and/or be applied on the food product that can be blended, feed product or medicine and use, still, handle solid food products Surface for example should also be as being effective by the inside of injection treatment this kind of product.Also in other embodiments, described group Compound can be used as marinade (marinade), breading (breading), wipe flavoring (seasoning rub), hanging (glaze), coloring agent mixture etc. is applied, crucial standard be subjected to the surface of bacterial degradation and yeast and mould contamination can be with Touch the antifungal composition.Also in other embodiments, by by composition be applied to packaging for foodstuff and then By packaging applications in food surface, the composition can be contacted with food surface indirectly.The optimised quantity to be used will Depending on by the composition of certain food product to be processed and method for composition to be applied to food surface, but can lead to Cross simple experiment determination.
In a further preferred embodiment, methods described includes one or more fermentation steps, and at least one Lactobacillus paracasei bacterial strain or the antifungal composition can quilts before, during or after one or more fermentation steps herein It is added in food product, feed product or medicine.
Preferably, methods described be included in exist it is at least one according to the present invention Lactobacillus paracasei bacterial strain in the case of With a period of time fermentation substrate, such as milk substrate, the time is sufficient so that at least one secondary cheese according to the present invention Lactobacillus strain shows antifungal activity.This antifungal activity, which has, suppresses yeast and/or mould with least one The effect grown in the product of strain fermentation.
In one even more preferably embodiment, methods described includes using starter culture cultured milk's substrate, described Starter culture, which includes, to be selected from at least one bacterial strain of subordinate：Lactobacillus, streptococcus, lactococcus and leukonid Category, such as at least one lactobacillus bulgaricus (Lactobacillus bulgaricus) bacterial strain and at least one thermophilus Bacteria strain (Streptococcus thermophilus), or such as at least one Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis) bacterial strain, at least one leuconostoc mesenteroides sub species cremoris (Leuconostoc mesenteroides subsp.cremoris) bacterial strain and at least one Lactococcus lactis biacetyl subspecies (Lactococcus lactis subsp.diacetylactis) bacterial strain.
The fifth aspect of the present invention is related to the food as obtained by method described according to the fourth aspect of the invention and produced Product.
The sixth aspect of the present invention is related to according to the Lactobacillus paracasei bacterial strain of first aspect or according to the anti-of second aspect Epiphyte composition is used for the purposes for preparing food product, feed product or medicine.Preferably, by using according to first aspect Lactobacillus paracasei bacterial strain or the food product produced according to the antifungal composition of second aspect are cheese, such as Ka Teji Cheese, it is luxuriant and rich with fragrance up to cheese, cut up to cheese, Palma cheese, cheese, angstrom door tal fibre cheese, red rich cheese, up to milk draw in Surrey Cheese in junket, Ai Danmu cheese, Fei Dashi cheese, blue cheese, halogen cheese, card awns Bel cheese or cloth.
The last aspect of the present invention is related to according to the Lactobacillus paracasei bacterial strain of first aspect or according to the anti-of second aspect Epiphyte composition is used for the purposes for suppressing the growth of yeast and mould, especially in food product and feed product.
Embodiment of the present invention is hereafter described by non-limiting example.
Embodiment
Embodiment 1：The Attack Research carried out with Lactobacillus paracasei CHCC14676 on Yoghourt
In order to which visual examination Lactobacillus paracasei bacterial strain CHCC14676 is for different moulds --- it is penicillium brevicompactum, common Mould, aspergillus versicolor and light spore mould --- inhibition, be prepared for containing 1.5% fat Yoghourt：
Water-bath is heat-treated homogeneous milk (1.5% fat) 5 minutes in 1L bottle at 95 DEG C ± 1 DEG C, and cools down immediately.With 0.02% inoculation Commercial starter cultures (the F-DVS YF-L901 that can be obtained from the Chr.Hansen A/S of Denmark).With HOLDBACTMYM-B (20DCU/100L) or Lactobacillus paracasei CHCC14676 (1 × 107CFU/ml milk) is further inoculated with, and And a bottle is used as with reference to and is only inoculated with starter culture.
The cultured milk at 43 DEG C ± 1 DEG C is until pH reaches 4.60 ± 0.1.Obtained Yoghourt is poured into cup (100g) And stored at 7 DEG C ± 1 DEG C.
After Yoghourt is prepared one day, it is inoculated into duplicate in equipment for prepn. of yoghurt using different moulds as surface contaminant, A bacterial plaque is formed on Yoghourt surface, target is 100 spore/bacterial plaques.Visually rank is mould after being stored 45 days at 7 DEG C ± 1 DEG C The growth of bacterium.
The result of Yoghourt test is presented in Fig. 1, and Fig. 1 shows penicillium brevicompactum (M1), penicillium communne (M6), aspergillus versicolor (M7) (gone on top) or on Yoghourt prepared by the milk by being fermented using only starter culture YF-L901 by making with light spore mould (M8) With starter culture and HOLDBACTM(center row) all grows well on Yoghourt prepared by the milk of YM-B cultures fermentation.Phase Than under, when Lactobacillus paracasei CHCC14676 is present in milk fermentation process (bottom row), the growth of the mould of all tests All it is inhibited by.
Embodiment 2：The quantitative determination of the inhibition of the Lactobacillus paracasei CHCC14676 confrontation inferior Dbaly yeasts of the Chinese
In order to which quantitative testing Lactobacillus paracasei CHCC14676 is for the inhibition of the inferior Dbaly yeast of the Chinese, it is prepared for Yoghourt：
Water-bath is heat-treated homogeneous milk (1.5% fat) 5 minutes and cooled down immediately in 1L bottles at 95 DEG C ± 1 DEG C.With 0.02% inoculation Commercial starter cultures (the F-DVS YF-L901 that can be obtained from the Chr.Hansen A/S of Denmark).With HOLDBACTM YM-B(20DCU/100L)、HOLDBACTMYM-C (10DCU/100L) or Lactobacillus paracasei CHCC14676 (1 ×107CFU/g milk) is further inoculated with, and a bottle is used as with reference to and is only inoculated with starter culture.
The cultured milk at 43 DEG C ± 1 DEG C is until pH reaches 4.60 ± 0.1.Obtained Yoghourt is poured into cup (100g) And stored at 7 DEG C ± 1 DEG C.
After Yoghourt is prepared one day, it is inoculated with duplicate in cup with the yeast of 1.00ml/ cups, target 20CFU/g.Will Yeast is evenly dispersed into Yoghourt.Cup is closed the lid and is stored at 7 DEG C ± 1 DEG C, and in the following manner with it is suitable when Between interval analyzed for the pollution level of the inferior Dbaly yeast of the Chinese：By 1ml Yoghourts with other in salt solution peptone The 1 times of appropriate dilution plating prepared in (saline peptone) on yeast glucose chloramphenicol (YGC) agar, Then oxygen supply is incubated 5 days at 25 DEG C.
As illustrated in figure 2, when being inoculated with before fermentation together with starter culture YF-L901, secondary cheese be present In the case of lactobacillus strain CHCC14676, the growth of the inferior Dbaly yeast of the Chinese is suppressed.The bacterial strain is than commercially available culture HOLDBACTMYM-B and HOLDBACTMYM-C causes significantly higher suppression.
Embodiment 3：Single Lactobacillus paracasei CHCC14676 and Lactobacillus rhamnosus CHCC5366 and its combination confrontation The semiquantitative determination of the inhibition of different mould contamination things
In order to carry out half to single Lactobacillus paracasei CHCC14676 and Lactobacillus rhamnosus CHCC5366 and its combination Quantitative testing, analyzed using agar, simulate the manufacturing process and product of Yoghourt：
Homogeneous milk (1.5%w/v fat) is heat-treated at 95 DEG C 5 minutes, and cooled down immediately.With 0.02% commercially available of inoculation Sub- culture (the F-DVS YC-350 that can be obtained from the Chr.Hansen A/S of Denmark), and milk is assigned in 220ml bottles. With 1 × 107CFU/ml total concentration is further inoculated with Lactobacillus paracasei CHCC14676, rhamnose breast bar respectively in bottle The combination of bacterium CHCC5366 and both bacterial strains.Except the bottle that starter culture is not inoculated with further is used as joining According to.In addition, the bromocresol purple of addition 5% and bromocresol green pH indicator are used to refer to acidification rate in all bottles Show, and for making matrix obtain blue/green, this can more easily detect the subsequent growth for causing target yeast and mould. All bottles are all incubated at 43 ± 1 DEG C in a water bath, and are fermented under these conditions until pH reaches 4.60 ± 0.1.Hair After ferment, immediately cooled on ice bottle and acutely vibrate with crush coagulation.Then cultured milk is warming up to 40 DEG C of temperature And be added in 40ml 5% sterile letheen solution, the sterile letheen solution has melted and has been cooled to 60 DEG C.Then will The solution is poured into sterile petri dish, and dry flat board 30 minutes in LAF workbench.
By the selected mould suitably diluted receive ground mould (10 ×), penicillium communne (100 ×), aspergillus versicolor (100 ×) and Skin falls the spore suspension point sample grown completely of mould (100 ×) to flat board.Flat board is incubated and with suitable and rule at 7 DEG C The time interval of rule checks the growth of mould.
The result of agar analysis is presented in Fig. 3, and Fig. 3 shows the mould of all tests by being sent out using only starter culture (reference) all very well grows on agar plate made from the milk of ferment.However, work as Lactobacillus paracasei CHCC14676 or sandlwood When sugared lactobacillus CHCC5366 is present in milk fermentation process, obtained flat board consumingly reduces the growth of all moulds.In addition, When Lactobacillus paracasei CHCC14676 and Lactobacillus rhamnosus CHCC5366 combinations are present in milk fermentation process, it was observed that Fall the even more strong suppression of mould particularly with the penicillium communne on point sample to flat board, aspergillus versicolor and skin.
Embodiment 4：Single Lactobacillus paracasei CHCC14676 and Lactobacillus rhamnosus CHCC14226 and its combination pair The semiquantitative determination of the inhibition of anti-different mould contamination thing
In order to be carried out to single Lactobacillus paracasei CHCC14676 and Lactobacillus rhamnosus CHCC14226 and its combination Sxemiquantitative is examined, and is analyzed using agar, is simulated the manufacturing process and product of Yoghourt：
Homogeneous milk (1.5%w/v fat) is heat-treated at 95 DEG C 5 minutes, and cooled down immediately.With 0.02% commercially available of inoculation Sub- culture (the F-DVS that can be obtained from the Chr.Hansen A/S of DenmarkMild 220ml bottles), and by milk are assigned to In son.With 1 × 107CFU/ml total concentration is further inoculated with Lactobacillus paracasei CHCC14676, rhamnose respectively in bottle The combination of lactobacillus CHCC14226 and both bacterial strains.A bottle quilt not being inoculated with further except starter culture As reference.In addition, the bromocresol purple of addition 5% and bromocresol green pH indicator are used to enter acidification rate in all bottles Row instruction, and for making matrix obtain blue/green, this can more easily examine the subsequent growth for causing target yeast and mould Survey.All bottles are all incubated at 43 ± 1 DEG C in a water bath, and are fermented under these conditions until pH reaches 4.60 ± 0.1. After fermentation, immediately cooled on ice bottle and acutely vibrate with crush coagulation.Then cultured milk is warming up to 40 DEG C of temperature Spend and be added in 40ml 5% sterile letheen solution, the sterile letheen solution has melted and has been cooled to 60 DEG C.Then The solution is poured into sterile petri dish, and dry flat board 30 minutes in LAF workbench.
By the selected mould suitably diluted receive ground mould (10 ×), penicillium communne (100 ×), aspergillus versicolor (100 ×) and Skin falls the spore suspension point sample grown up completely of mould (100 ×) to flat board.Flat board is incubated and with suitable and rule at 7 DEG C The time interval of rule checks the growth of mould.
The result of agar analysis is presented in Fig. 4, and Fig. 4 shows the mould of all tests by being sent out using only starter culture All very well grown on agar plate (reference) made from the milk of ferment.However, work as Lactobacillus paracasei CHCC14676 or sandlwood When sugared lactobacillus CHCC14266 is present in milk fermentation process, obtained flat board consumingly reduces the growth of all moulds.In addition, When Lactobacillus paracasei CHCC14676 and Lactobacillus rhamnosus CHCC14266 combinations are present in milk fermentation process, it was observed that Fall the even more strong suppression of mould for the penicillium communne on point sample to flat board, aspergillus versicolor and skin.
Embodiment 5：With the Lactobacillus rhamnosus CHCC5366 and Lactobacillus paracasei CHCC14676 of combination in Greek style acid The Attack Research carried out on milk
The Lactobacillus rhamnosus CHCC5366 and Lactobacillus paracasei CHCC14676 combined for visual examination is for Mucor The inhibition of category, Greek style Yoghourt is substantially prepared as follows：
With Commercial starter cultures (F-DVSYF-L901 that can be obtained from the Chr.Hansen A/S of Denmark) with 0.02% It is inoculated with 1.5% fat milk of pasteurization.Only it is used as reference with a collection of of starter culture inoculation.Another batch is further used sandlwood Sugared lactobacillus strain CHCC5366 (5 × 106) and Lactobacillus paracasei bacterial strain CHCC14676 (5 × 10 CFU/g6CFU/g group) Splice grafting kind.
Cultured milk is to final pH 4.55 (6-7 hours) at 43 DEG C ± 1 DEG C.Then Yoghourt is cooled to 2 bars of back-pressure 25 DEG C ± 1 DEG C and it is stored in 6 DEG C ± 1 DEG C.
Prepared one day after in Yoghourt, by applying a bacterial plaque on Yoghourt surface, using mucor as surface contaminant one Two parts of ground of formula are inoculated into equipment for prepn. of yoghurt, and target inoculum density is 100 spore/bacterial plaques.Mesh after being stored 15 days at 22 DEG C ± 1 DEG C The growth of mould is estimated in test and appraisal.
The result of Greek style Yoghourt test is presented in Fig. 5, and Fig. 5 shows mucor by using only starter culture Grown well on Yoghourt made from the milk of (left cup) fermentation.By contrast, as Lactobacillus rhamnosus CHCC5366 and secondary cheese When lactobacillus CHCC14676 is present in milk fermentation process (right cup), the growth of mucor is suppressed.
Embodiment 6：With Lactobacillus rhamnosus CHCC5366 and Lactobacillus paracasei CHCC14676 combination in Greek style acid Second of Attack Research on milk
In order to visual examination Lactobacillus rhamnosus CHCC5366 and Lactobacillus paracasei CHCC14676 combination for black root The inhibition of mould (black bread mold) rhizopus stolonifer, Greek style Yoghourt is substantially prepared as follows：
With Commercial starter cultures (F-DVSYF-L901 that can be obtained from the Chr.Hansen A/S of Denmark) with 0.02% It is inoculated with 1.5% fat milk of pasteurization.Use HOLDBACTMYM-B (10DCU/100L), use Lactobacillus rhamnosus strain CHCC5366(1×107CFU/g), or with Lactobacillus rhamnosus strain CHCC5366 (5 × 106) and Lactobacillus paracasei CFU/g Bacterial strain CHCC14676 (5 × 106CFU/g combination) is further inoculated with milk.It is a collection of to be used as with reference to and only connect with starter culture Kind.
Cultured milk is to final pH 4.55 (6-7 hours) at 43 DEG C ± 1 DEG C.Then Yoghourt is cooled to 2 bars of back-pressure 25 DEG C ± 1 DEG C and it is stored in 6 DEG C ± 1 DEG C.
Prepared one day after in Yoghourt, by one bacterial plaque in way on Yoghourt surface, using rhizopus stolonifer as surface contaminant one Two parts of ground of formula are inoculated into equipment for prepn. of yoghurt, and target stains are 100 spore/bacterial plaques.Estimate and comment after being stored 42 days at 7 DEG C ± 1 DEG C Estimate the growth of mould.
The result of Greek style Yoghourt test is presented in Fig. 6, and Fig. 6 shows rhizopus stolonifer by using only starter culture On Yoghourt made from the milk of fermentation (" R ", i.e. reference) or by using starter culture and HOLDBACTMYM-B cultures (YM- B) grown well on Yoghourt made from the milk of fermentation.However, in the presence of Lactobacillus rhamnosus CHCC5366 (5366), branch of crawling The growth of head mold is suppressed significantly.When Lactobacillus rhamnosus CHCC5366 and Lactobacillus paracasei CHCC14676 are present in milk When in fermentation process (5366+14676), the growth of rhizopus stolonifer is almost totally constrained.
Embodiment 7：With the Lactobacillus paracasei CHCC14676 and Lactobacillus rhamnosus CHCC12697 of combination in sour cream On Attack Research
In order to visual examination Lactobacillus paracasei CHCC14676 and Lactobacillus rhamnosus CHCC12697 combination for not The inhibition of the growth of same mould, is substantially prepared for sour cream as follows：
(can be from Denmark with the commercially available heterofermentation starter culture of 0.01% inoculation in the high fat milk of pasteurization The F-DVS DSG-2000 that Chr.Hansen A/S are obtained).Use HOLDBACTMYM-B (10DCU/100L) or with secondary cheese breast bar Bacterium CHCC14676 (2.5 × 106) and Lactobacillus rhamnosus CHCC12697 (2.5 × 10 CFU/g6CFU/g combination) further connects Kind milk.It is a collection of to be used as with reference to and be only inoculated with starter culture.
Cultured milk reaches 4.60 ± 0.05 until pH at 28 DEG C ± 1 DEG C, and sour cream is post-processed.Whisk yoghout Oil is simultaneously cooled to 20 DEG C ± 1 DEG C and stored at 7 DEG C ± 1 DEG C.
It is dirty using different moulds as surface by applying a bacterial plaque in Yoghourt oil meter face after sour cream is prepared one day Dye thing is inoculated into Yoghourt lubricating cup in duplicate, and aimed concn is 100 spore/bacterial plaques.Stored 61 days at 7 DEG C ± 1 DEG C The growth of visually rank mould afterwards.
The result of sour cream test is presented in Fig. 7, and Fig. 7 shows penicillium brevicompactum (M1), penicillium communne (M6), variegated song Mould (M7) and light spore mould (M8) is on sour cream made from the milk as being fermented using only starter culture (top row) or by using Starter culture and HOLDBACTMAll grown well on sour cream (center row) made from the milk of YM-B cultures fermentation.Phase Than under, when being present in Lactobacillus paracasei CHCC14676 and Lactobacillus rhamnosus CHCC12697 in milk fermentation process (bottom row), the growth of the mould of all tests are all inhibited by.
Embodiment 8：Lactobacillus paracasei CHCC14676 and Lactobacillus rhamnosus CHCC12697 combination confrontation sour cream In the inferior Dbaly yeast of the Chinese inhibition quantitative determination.
For quantitative testing Lactobacillus paracasei bacterial strain CHCC14676 and Lactobacillus rhamnosus CHCC12697 combination pair The inhibition of the anti-inferior Dbaly yeast of the Chinese, is substantially prepared for sour cream as follows：
(can be from Denmark with the commercially available heterofermentation starter culture of 0.01% inoculation in the high fat milk of pasteurization The F-DVS DSG-2000 that Chr.Hansen A/S are obtained).Use HOLDBACTMYM-B (10DCU/100L) or with secondary cheese breast bar Bacterium CHCC14676 (2.5 × 106) and Lactobacillus rhamnosus CHCC12697 (2.5 × 10 CFU/g6CFU/g combination) further connects Kind milk.It is a collection of to be used as with reference to and be only inoculated with starter culture.
Cultured milk reaches 4.60 ± 0.05 until pH at 28 DEG C ± 1 DEG C, and sour cream is post-processed.Whisk yoghout Oil is simultaneously cooled to 20 DEG C ± 1 DEG C and stored at 7 DEG C ± 1 DEG C.
After sour cream is prepared one day, the yeast of 1.00 milliliters/glass is inoculated into cup in duplicate, target is 20CFU/g.Yeast is evenly dispersed in sour cream.Cup is closed the lid and is stored in 7 DEG C ± 1 DEG C, and by with lower section Formula is analyzed for the pollution level of the inferior Dbaly yeast of the Chinese at appropriate time intervals：By 1 milliliter of sour cream and in addition The 1 times of appropriate dilution plating prepared in salt solution peptone is on yeast glucose chloramphenicol (YGC) agar, then Oxygen supply is incubated 5 days at 25 DEG C.
As illustrated in figure 8, when being inoculated with before fermentation together with starter culture DSG-2000, secondary cheese be present In the case of lactobacillus CHCC14676 and Lactobacillus rhamnosus CHCC12697 combination, the growth of the inferior Dbaly yeast of the Chinese by Suppress.The bacterial strain is than commercially available culture HOLDBACTMYM-B causes significantly higher suppression.
Embodiment 9：With the Lactobacillus rhamnosus CHCC5366 and Lactobacillus paracasei CHCC14676 of combination in white halogen cheese On Attack Research
For Lactobacillus rhamnosus CHCC5366 and Lactobacillus paracasei CHCC14676 the confrontation wine brewing of quantitative testing combination The inhibition of yeast, it is prepared for white halogen cheese：
With Commercial starter cultures (the FD-DVS SafeIT-1 that can be obtained from the Chr.Hansen A/S of Denmark) with every 1000 liters of (l) milk 40U are inoculated with the milk of pasteurization and standardization.Use HOLDBACTMYM-B (10DCU/100L) uses rhamnose Lactobacillus strain CHCC5366 (5 × 106) and Lactobacillus paracasei bacterial strain CHCC14676 (5 × 10 CFU/g6CFU/g combination) Further it is inoculated with milk.It is a collection of to be used as with reference to and be only inoculated with starter culture.At 36 DEG C ± 1 DEG C, using Chy-Max plus (from The Chr.Hansen A/S of Denmark are obtained) 90 points of rennet processing is carried out to milk with every 220 milliliters of 1000 liters (l) (ml) Clock, then cut.In pH 6.0, by curdled milk with simultaneously discharge opeing is pumped into mould, to reach final pH 4.8-4.7 (20-24 hours).Cheese is added in the tank containing cold brine (8%) and stored at 5 DEG C ± 1 DEG C.
Prepare one day after, yeast is inoculated into the tank in duplicate, target 20CFU/ml in white halogen cheese.Will Yeast is evenly dispersed in bittern.Cup is closed the lid and is stored at 7 DEG C ± 1 DEG C, for up to 40 days, and by with lower section Formula is at appropriate time intervals for the pollution level analysis 10g of saccharomyces cerevisiae：Appropriate 1 that will be prepared in salt solution peptone Times dilution plating is on yeast glucose chloramphenicol (YGC) agar, and then oxygen supply is incubated 5 days at 25 DEG C.
As illustrated in figure 9, when being inoculated with together with starter culture SafeIT-1, in the rhamnose breast bar that combination be present In the case of bacterium CHCC5366 and Lactobacillus paracasei CHCC14676, the growth of saccharomyces cerevisiae is suppressed.The bacterial strain is than commercially available Culture HOLDBACTMYM-B causes significantly higher suppression.
Embodiment 10：With the Lactobacillus rhamnosus CHCC5366 and Lactobacillus paracasei CHCC14676 of combination in white halogen milk Second of Attack Research on junket
In order to which the Lactobacillus rhamnosus CHCC5366 and Lactobacillus paracasei CHCC14676 of quantitative testing combination resist mark The inhibition of this kluyveromyces, as the description in embodiment 1 is prepared for white halogen cheese, the exception is that the tank uses horse Gram this kluyveromyces are inoculated with as yeast pollutant：
As illustrated in Figure 10, when being inoculated with together with starter culture SafeIT-1, in the rhamnose breast that combination be present In the case of bacillus CHCC5366 and Lactobacillus paracasei CHCC14676, the growth of kluyveromyces marxianus is suppressed.It is described Bacterial strain is than commercially available culture HOLDBACTMYM-B causes significantly higher suppression.
Embodiment 11：With the Lactobacillus rhamnosus CHCC5366 and Lactobacillus paracasei CHCC14676 of combination in white halogen milk Third time Attack Research on junket
It is common for Lactobacillus rhamnosus CHCC5366 and Lactobacillus paracasei the CHCC14676 confrontation of visual examination combination The inhibition of mould, as the description in embodiment 1 is prepared for white halogen cheese：
After white halogen cheese is prepared 7 days, cheese is removed from bittern, and using penicillium communne as surface contaminant one Two parts of ground of formula are inoculated on cheese, three bacterial plaques are formed on cheese surface, target is 100 spore/bacterial plaques.At 7 DEG C ± 1 DEG C Lower storage 12 days and after then being stored 16 days at 12 DEG C ± 1 DEG C, the life of the different penicillium communne separation strains of visually rank two It is long.
The result of white halogen cheese test is presented in Figure 11, Figure 11 show two penicillium communne separation strains by using only Grown well on white halogen cheese (left side) made from the milk of starter culture inoculation.By contrast, Lactobacillus rhamnosus is worked as When CHCC5366 and Lactobacillus paracasei CHCC14676 are present in cheese production process (right side), the growth of penicillium communne is strong Strong suppression.
Embodiment 12：With attacking for the confrontation Pan Shi moulds of Lactobacillus rhamnosus CHCC5366 and CHCC14676 on Yoghourt Hit research
For the single Lactobacillus rhamnosus strain CHCC5366 of visual examination or itself and Lactobacillus paracasei CHCC14676 The inhibition for mould Pan Shi moulds is combined, is prepared for the Yoghourt containing 1.5% fat：
Water-bath is heat-treated homogeneous milk (1.5% fat) 5 minutes in 1L bottle at 95 DEG C ± 1 DEG C, and cools down immediately.With 0.02% inoculation Commercial starter cultures (the F-DVS YF-L901 that can be obtained from the Chr.Hansen A/S of Denmark).With HOLDBACTMYM-B (20DCU/100L), individually use Lactobacillus rhamnosus CHCC5366 (1 × 107CFU/ml), or rhamnose is used Lactobacillus CHCC5366 (5 × 106CFU/ml) with Lactobacillus paracasei CHCC14676 (5 × 106CFU/ml combination) is further It is inoculated with milk.One bottle is used as with reference to and is only inoculated with starter culture.
The cultured milk at 43 DEG C ± 1 DEG C is until pH reaches 4.60 ± 0.1.Obtained Yoghourt is poured into cup (100g) simultaneously Stored at 7 DEG C ± 1 DEG C.
After Yoghourt is prepared one day, it is inoculated into duplicate in equipment for prepn. of yoghurt using different moulds as surface contaminant, A bacterial plaque is formed on Yoghourt surface, target is 100 spore/bacterial plaques.Visually rank is mould after being stored 28 days at 7 DEG C ± 1 DEG C The growth of bacterium.
The result of Yoghourt test is presented in Figure 12, and Figure 12 shows Pan Shi moulds by using only starter culture YF- Made from the milk of L901 fermentations on Yoghourt or by using starter culture and HOLDBACTMThe milk of YM-B cultures fermentation is made Yoghourt on all grow well.By contrast, when Lactobacillus rhamnosus CHCC5366 is present in milk fermentation process, Pan Shi The growth of mould is reduced strongly, moreover, Lactobacillus rhamnosus CHCC5366 and Lactobacillus paracasei CHCC14676 combination cause The growth inhibition of even more strong Pan Shi moulds.
Embodiment 13：With Lactobacillus rhamnosus CHCC5366 and CHCC14676 under different storage temperatures on Yoghourt Attack Research
For the single Lactobacillus rhamnosus strain CHCC5366 of visual examination or itself and Lactobacillus paracasei CHCC14676 Combination falls the inhibition of mould under different storage temperatures for mould skin, is prepared for the Yoghourt containing 1.5% fat：
Water-bath is heat-treated homogeneous milk (1.5% fat) 5 minutes in 1L bottle at 95 DEG C ± 1 DEG C, and cools down immediately.With 0.02% inoculation Commercial starter cultures (the F-DVS YF-L901 that can be obtained from the Chr.Hansen A/S of Denmark).With HOLDBACTMYM-B (20DCU/100L), individually use Lactobacillus rhamnosus CHCC5366 (1 × 107CFU/ml), or rhamnose is used Lactobacillus CHCC5366 (5 × 106CFU/ml) with Lactobacillus paracasei CHCC14676 (5 × 106CFU/ml combination) is further It is inoculated with milk.One bottle is used as with reference to and is only inoculated with starter culture.
The cultured milk at 43 DEG C ± 1 DEG C is until pH reaches 4.60 ± 0.1.Obtained Yoghourt is poured into cup (100g) simultaneously Stored at 7 DEG C ± 1 DEG C.
After Yoghourt is prepared one day, skin is fallen into mould and is inoculated into duplicate in equipment for prepn. of yoghurt as surface contaminant, A bacterial plaque is formed on Yoghourt surface, target is 100 spore/bacterial plaques.At 7 DEG C ± 1 DEG C, 12 DEG C ± 1 DEG C or 22 DEG C ± 1 DEG C The growth of visually rank mould after storing 36 days.
The result of Yoghourt test is presented in Figure 13, and Figure 13 shows that skin falls mould by using only starter culture YF- Made from the milk of L901 fermentations on Yoghourt or by using starter culture and HOLDBACTMThe milk of YM-B cultures fermentation is made Yoghourt on all grown well under all storage temperatures.By contrast, when Lactobacillus rhamnosus CHCC5366 individually or with When Lactobacillus paracasei CHCC14676 combinations are present in milk fermentation process, stored 36 days at 7 DEG C ± 1 DEG C and 12 DEG C ± 1 DEG C In the case of two kinds, the growth that skin falls mould is completely blocked.In the case of 22 DEG C of ± 1 DEG C of storages, skin falls being grown in for mould Consumingly reduced in the case of Lactobacillus rhamnosus CHCC5366 being present, and Lactobacillus rhamnosus CHCC5366 and pair be present Lactobacillus casei CHCC14676 is even more strongly reduced in the case of combining.
Preserved material and Expert Solutions
Applicant requires that before the grant date of this patent the sample of the conserving microorganism of following notice is only available for professional people Scholar uses.
Lactobacillus paracasei bacterial strain CHCC14676 was deposited in German microorganism on 2 2nd, 2012 and cell culture is protected Tibetan center (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH；DSMZ) (Inhoffenstr.7B, D-38124Braunschweig) and it is designated deposit number DSM25612.
Lactobacillus rhamnosus strain CHCC5366 was deposited in German microorganism and cell culture on October 14th, 2009 Collection (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH；DSMZ) (Inhoffenstr.7B, D-38124Braunschweig) and it is designated deposit number DSM23035.
Lactobacillus rhamnosus strain CHCC12697 is deposited in German microorganism on March 1st, 2011 and cell culture is protected Tibetan center (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH；DSMZ) (Inhoffenstr.7B, D-38124Braunschweig) and it is designated deposit number DSM24616.
Lactobacillus rhamnosus strain CHCC14226 was deposited in German microorganism and cell culture on March 15th, 2011 Collection (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH；DSMZ) (Inhoffenstr.7B, D-38124Braunschweig) and it is designated deposit number DSM24562.
Preserved material was prepared according to " international recognition is used for the microbial preservation budapest treaty of proprietary program ".
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Kosikowski, F.V. and Mistry, V.V., " Cheese and Fermented Milk Foods ",
1997, the third edition, F.V.Kosikowski, L.L.C.Westport, CT

Claims (20)

1. a kind of Lactobacillus paracasei (Lactobacillus paracasei) bacterial strain, it is to be protected with deposit number DSM25612 It is hidden in the Lactobacillus paracasei bacterial strain CHCC14676 of German microorganism and Cell Culture Collection.
2. a kind of antifungal composition, it includes Lactobacillus paracasei bacterial strain according to claim 1.
3. a kind of antifungal composition, it includes Lactobacillus paracasei bacterial strain according to claim 1 and at least one mouse Lee's sugar lactobacillus (Lactobacillus rhamnosus) bacterial strain.
4. antifungal composition according to claim 3, wherein at least one Lactobacillus rhamnosus strain be selected from by Group consisting of：The mouse that Lactobacillus rhamnosus CHCC12697 that deposit number is DSM24616, deposit number are DSM24652 Lee sugar lactobacillus CHCC14226 and Lactobacillus rhamnosus CHCC5366 that deposit number is DSM23035.
5. a kind of food product, its include Lactobacillus paracasei bacterial strain according to claim 1 or according to claim 2 to Antifungal composition described in any one of 4.
6. food product according to claim 5, it includes the amount for effectively assigning the food product antifungal property Lactobacillus paracasei bacterial strain according to claim 1.
7. according to the food product described in claim 5 or claim 6, wherein the food product is selected from what is consisted of Group：Fruit and fruit derived product, vegetables and vegetables derived product, cereal and cereal derived product, dairy products and meat and its Mixture.
8. according to the food product described in claim 5 or claim 6, wherein the food product is selected from what is consisted of Group：Poultry and marine products class.
9. food product according to claim 7, wherein the food product is dairy products, and the dairy products are medium temperature Fermented-milk products or hot fermentation dairy products.
10. food product according to claim 9, wherein the dairy products are Yoghourt or sour cream.
11. a kind of feed product, it includes Lactobacillus paracasei bacterial strain according to claim 1 or according to claim 2 To the antifungal composition described in any one of 4.
12. a kind of medicine, it includes Lactobacillus paracasei bacterial strain according to claim 1 or according in claim 2 to 4 Any one described in antifungal composition.
13. a kind of method for manufacturing food product, feed product or medicine, it includes in the fabrication process will be according to claim Lactobacillus paracasei bacterial strain described in 1 or the antifungal composition according to any one of claim 2 to 4 are added to institute State in food product, feed product or medicine, and control Fabrication parameter in the fabrication process so that the Lactobacillus paracasei bacterium The concentration of strain keeps constant or improved.
14. according to the method for claim 13, wherein the concentration of the Lactobacillus paracasei bacterial strain is food production described in per g Product, feed product or medicine at least 1 × 106Food product, feed product or medicine at least 1 described in cfu or respectively every ml × 106Cfu, or per cm2The food product, the surface at least 1 × 10 of feed product or medicine5cfu。
15. according to the method for claim 13, it includes：
(a) added during the food product, feed product or medicine is manufactured according to any in claim 2 to 4 Antifungal composition described in so that at least one Lactobacillus rhamnosus strain and/or the Lactobacillus paracasei bacterium The concentration of strain is respectively food product, feed product or medicine at least 1 × 10 described in per g6Cfu or per food product described in ml, Feed product or medicine at least 1 × 106Cfu, or per cm2The food product, feed product or medicine surface at least 1 × 105cfu；With
(b) Fabrication parameter is controlled in the fabrication process so that at least one Lactobacillus rhamnosus strain and/or the pair are dry The concentration of Lactobacillus paracasei bacterial strain improves or keeps constant.
Walked 16. the method according to any one of claim 13 to 15, wherein methods described include one or more fermentations Suddenly.
17. according to the method for claim 16, wherein methods described includes using starter culture cultured milk's substrate, described Sub- culture, which includes, to be selected from at least one bacterial strain of subordinate：Lactobacillus (Lactobacillus) category, streptococcus (Streptococcus) category, galactococcus (Lactococcus) category and leukonid (Leuconostoc) category.
18. Lactobacillus paracasei bacterial strain according to claim 1 or according to any one of claim 2 to 4 Antifungal composition is used for the purposes for preparing food product, feed product or medicine.
19. Lactobacillus paracasei bacterial strain according to claim 1 or according to any one of claim 2 to 4 Antifungal composition is used for the non-therapeutic use for suppressing the growth of fungi and mould.
20. Lactobacillus paracasei bacterial strain according to claim 1 or according to any one of claim 2 to 4 Purposes of the antifungal composition in the medicine for preparing the growth for being used for suppressing fungi and mould.
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